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SETTING: Tiruvallur District, South India, where one baseline tuberculosis (TB) disease prevalence survey followed by three repeat prevalence surveys were conducted every 2.5 years between 1999 and 2008, and where the DOTS strategy was implemented in 1999. OBJECTIVE: To rule out the possibility that the observed decline in TB prevalence was influenced by conducting repeat prevalence surveys, we compared the findings from two surveys: the third repeat survey conducted in 2006-2008 and an independent single survey in a neighbouring area conducted in 2008-2009. DESIGN: An independent survey was conducted to estimate the prevalence of TB in the same district in 2008-2009 using a different set of villages and employing repeat survey methodology. The independent survey findings were compared with those of the third repeat survey. RESULTS: The estimated prevalence rate of culture- and smear-positive TB was respectively 401 per 100 000 and 186 per 100 000 population in the third repeat survey area. The corresponding rates were 340 and 184/100 000 in the independent survey area. The difference in prevalence was not significant (culture P = 0.09; smear P = 0.93). CONCLUSION: The estimated prevalence rates in the two different sample survey areas were comparable, indicating that the repeated prevalence surveys in the study area did not influence the observed decline in TB disease prevalence.

Background & objectives: Increase in the isolation of drug resistant phenotypes of Mycobacterium tuberculosis necessitates accuracy in the testing methodology. Critical concentration defining resistance for ethionamide (ETO), needs re-evaluation in accordance with the current scenario. Thus, re-evaluation of conventional minimum inhibitory concentration (MIC) and proportion sensitivity testing (PST) methods for ETO was done to identify the ideal breakpoint concentration defining resistance. Methods: Isolates of M. tuberculosis (n=235) from new and treated patients were subjected to conventional MIC and PST methods for ETO following standard operating procedures. Results: With breakpoint concentration set at 114 and 156 µg/ml, an increase in specificity was observed whereas sensitivity was high with 80 µg/ml as breakpoint concentration. Errors due to false resistant and susceptible isolates were least at 80 µg/ml concentration. Interpretation & conclusions: Performance parameters at 80 µg/ml breakpoint concentration indicated significant association between PST and MIC methods.

BACKGROUND: Blinded rechecking of auramine-stained acid-fast bacilli (AFB) sputum smears using fluorescence microscopy (FM), especially FM using light-emitting diode (LED), is not well understood. OBJECTIVE: To examine the rechecking of auramine-stained sputum smears without restaining within a month using LED FM. METHODS: A total of 4799 centrifuged smears of sputum samples were stained by the auramine phenol method and examined using LED FM; 564 systematically selected smears were subjected to blinded rechecking without restaining by controllers. The initial results of the readers were compared to those of the controllers. Discrepancies were resolved by a referee. The quality of LED FM was assessed by the referee using the culture result as gold standard. RESULTS: Among the rechecked smears, one high false-negative error was made by a reader, while one high false-positive error and 19 high false-negative errors were made by the controllers. The errors were resolved by culture. Smear results for 18 slides were not available due to AFB fading. CONCLUSION: AFB colour fading using LED FM, which affected the accurate evaluation of blinded rechecking of AFB smears without restaining within a month, is confirmed in this large study.

BACKGROUND: The Revised National TB Control Program bases diagnosis of tuberculosis (TB) on sputum smear examination and response to a course of antibiotics, whereas World Health Organization recommends early chest radiography [chest x-ray (CXR)] for HIV-infected symptomatic patients. We evaluated the utility of initial CXR in the diagnostic algorithm for symptomatic HIV-infected patients with negative sputum smears. METHODS: HIV-infected ambulatory patients with cough or fever of ≥2 weeks and 3 sputum smears negative for acid-fast bacilli were enrolled in Chennai and Pune, India, between 2007 and 2009. After a CXR and 2 sputum cultures, a course of broad-spectrum antibiotics was given and patients were reviewed after 14 days. Sensitivity, specificity, positive and negative predictive values of symptoms, CXR, and various combinations for diagnosing pulmonary tuberculosis (PTB) were determined, using sputum culture as gold standard. RESULTS: Five hundred four patients (330 males; mean age: 35 years; median CD4: 175 cells per cubic millimeter) were enrolled. CXR had a sensitivity and specificity of 72% and 57%, respectively, with positive predictive value (PPV) of 21% and negative predictive value (NPV) of 93% to diagnose PTB. TB culture was positive in 49 of 235 patients (21%) with an abnormal initial CXR and 19 of 269 patients (7%) with a normal CXR (P

During the course of the anti-infective drug discovery programme, actinomycete strain D25 was recovered from the Thar Desert soil, Rajasthan, India. Actinomycin type of compound isolated from the strain D25 showed promising activity against multi drug resistant and extensively drug resistant M. tuberculosis isolates. The present study reports the characteristics and phylogenetic status of the actinomycete strain D25. Phenotypic and cell wall characteristics revealed that the strain belongs to the genus Streptomyces. Further 16s rRNA analysis confined the genus Streptomyces with 97% similarity to the closely related species Streptomyces althioticus KCTC 9752. The 16s rRNA sequence was submitted to GenBank with the accession number JN604533.1. According to Bossard et al. (2003) strain D25 was found to be a novel species of the genus Streptomyces from Thar Desert soil, Rajasthan.

Background Documentation of structured quality indicators for mycobacteriology laboratories supporting exclusively controlled clinical trials in pulmonary tuberculosis (PTB) is lacking. Objective To document laboratory indicators for a solid (Lowenstein–Jensen medium) culture system in a mycobacteriology laboratory for a period of 4 years (2007–2010). Methods The sputum samples, collected from PTB suspects/patients enrolled in clinical trials, were subjected to fluorescence microscopy, culture and drug sensitivity testing (DST). Data was retrospectively collected from TB laboratory registers and computed using pre-formulated Microsoft Office Excel. Laboratory indicators were calculated and analyzed. Results The number of samples processed in a calendar year varied from 6261 to 10,710. Of the samples processed in a calendar year, specimen contamination (4.8–6.9%), culture positives (78.4–85.1%) among smear positives, smear positives (71.8–79.0%) among culture positive samples, smear negatives among culture negative samples (95.2–96.7%), and average time to report DST results (76–97 days) varied as shown in parentheses. Conclusion Values of quality indicators in mycobacteriology laboratories supporting exclusively clinical trials of PTB have to be defined and used for meaningful monitoring of laboratories.

BACKGROUND: The performance of the Capilia test for rapid identification of Mycobacterium tuberculosis complex (MTC) in Mycobacterium Growth Indicator Tube (MGIT) positive samples with contaminating organisms is not well documented. OBJECTIVE: To assess the diagnostic yield of the Capilia test in the rapid identification of MTC in MGIT-positive cultures. DESIGN: A total of 459 selected sputum samples were cultured using BACTEC™ MGIT™ 960. Tubes flagged positive by the MGIT instrument (MGIT-positive) were examined for acid-fast bacilli and cording in smears, spotted on blood agar (BA), subcultured for biochemical tests and tested using the Capilia test. Based on smear and growth on BA, MGIT-positive tubes were grouped into MGIT true-positive, MGIT-positive with contamination and MGIT contamination. Performance parameters of Capiliatest such as sensitivity, specificity, efficiency, and positive and negative predictive values (PPV, NPV) for each of these groups were determined against biochemical tests as gold standard. RESULTS: Of the 346 MGIT-positives, respectively 233, 73 and 40 were MGIT true-positive, MGIT-positive with contamination and MGIT contamination. For the three groups, the PPV and NPV of the Capilia test were respectively 97%, 96% and 100%, and 32%, 27% and 60%. CONCLUSION: In settings with high contamination of MGIT cultures, the performance of the Capilia test is diminished.

Diagnosis of latent tuberculosis infection is a myth for want of a simple, direct tool. Simulation of hypoxic environment was done to create a novel hypothetical model for persistence using processed sputum samples. The adaptation of tubercle bacilli to hypoxic environment seems to be influenced by pre-existing clinical status of the patients at the time of sputum collection, resulting in varied growth pattern. Bacilli from 36 samples did not get adapted to latency of which 15 samples were from patients in whom the disease was well established and the tubercle bacilli in them probably did not experience any stress whatsoever. Similarly, 10 of the 37 samples showing the presence of cultivable cells in both aerobic and anaerobic conditions were from patients who had relapsed. The bacilli in these samples had been probably experiencing stress and thus were ready to adapt to the hypoxic environment. Diagnostic luciferase reporter phage assay for non-replicating persistors (DLRPA-NRP) identified 30 additional positives which failed to grow on Lowenstein–Jensen medium. Presence of viable bacilli in these samples was confirmed by reverse transcriptase-PCR (RT-PCR) for 16S rRNA indicating either the improved sensitivity of the assay to detect actively growing bacilli or its ability to detect non-replicating persistors. The utility of LRP assay to detect both dormant and active tubercle bacilli was explored in this work and was optimized using lysis inhibition to diagnose tuberculosis with rapidity, improved sensitivity and specificity. DLRPA-NRP, a rapid growth based assay is thus developed to detect both dormant and actively growing tubercle bacilli.

BACKGROUND: There is little information on the recovery of Mycobacterium tuberculosis complex from sputum samples collected in the field for bacteriological examination. SETTING: Tribal areas in the State of Madhya Pradesh, in central India. METHODS: Sputum specimens collected from subjects with chest symptoms in a disease prevalence survey were refrigerated until transportation to the laboratory. The specimens were processed for microscopy and culture. The samples were grouped based on the delay in processing for culture from the day of collection into three groups: 0-3, 4-7 and ≥8 days, and the recovery of M. tuberculosis complex in these groups was analysed. The statistical analysis was performed using χ(2) test. RESULTS: Of the 3651 processed specimens, 114 were positive for M. tuberculosis complex by culture and 96 by smear microscopy. The differences in the proportion of 'smear-positive, culture-positive', 'smear-positive, culture-negative' and 'smear-negative, culture-positive' samples between the three groups were not significant. CONCLUSION: In difficult-to-reach areas with limited resources, refrigeration of sputum specimens until they are transported for processing at a reference laboratory for culture seems not to significantly affect the recovery of M. tuberculosis complex isolates.

SummaryObjectivesTo estimate the prevalence of pulmonary tuberculosis (TB) disease amongst the Saharia, a ‘primitive’ tribe of Madhya Pradesh, Central India.MethodsA community-based cross-sectional TB prevalence survey was undertaken in the Saharia, a ‘primitive’ tribal community of Madhya Pradesh. A representative random sample of villages predominated by tribal populations was chosen from the selected block of Sheopur District. All eligible individuals were screened for chest symptoms related to TB. Sputum samples were collected from all eligible individuals, transported to the laboratory, and examined by Ziehl–Neelsen smear microscopy and solid media culture methods.ResultsOf the 11 468 individuals eligible for screening, 11 116 (96.9%) were screened for symptoms. The overall prevalence of pulmonary TB disease was 1518 per 100 000 population. Prevalence increased with age and the trend was statistically significant (p

Pyrazinamide (PZA) - an important drug in the anti-tuberculosis therapy, activated by an enzyme Pyrazinamidase (PZase). The basis of PZA resistance in Mycobacterium tuberculosis was owing to mutation in pncA gene coding for PZase. Homology modeling of PZase was performed using software Discovery Studio (DS) 2.0 based on the crystal structure of the PZase from Pyrococcus horikoshii (PDB code 1im5), in this study. The model comprises of one sheet with six parallel strands and seven helices with the amino acids Asp8, Asp49, Trp68, Lys96, Ala134, Thr135 and Cys138 at the active site. Five mutants were generated with Gly at position 8, Thr at position 96, Arg at position 104, Tyr and Ser at position 138. The Wild-type (WT) and five mutant models were docked with PZA. The results indicate that the mutants Lys96Thr, Ser104Arg Asp8Gly and Cys138Tyr may contribute to higher level drug resistance than Cys138Ser. These models provide the first in-silico evidence for the binding interaction of PZA with PZase and form the basis for rationalization of PZA resistance in naturally occurring pncA mutant strains of M. tuberculosis.

Background: Tuberculosis is curable, but community surveys documenting epidemiological impact of the WHO-recommended DOTS strategy on tuberculosis prevalence have not been published. We used active community surveillance to compare the impact of DOTS with earlier programmes. Methods: We conducted tuberculosis disease surveys using random cluster sampling of a rural population in South India approximately every 2.5 years from 1968 to 1986, using radiography as a screening tool for sputum examination. In 1999, DOTS was implemented in the area. Prevalence surveys using radiography and symptom screening were conducted at the start of DOTS implementation and after 2.5 years. Results: From 1968 to 1999, culture-positive and smear-positive tuberculosis declined by 2.3 and 2.5% per annum compared with 11.9 and 5.6% after DOTS implementation. The 2.5 year period of DOTS implementation accounted for one-fourth of the decline in prevalence of culture-positive tuberculosis over 33 years. Multivariate analysis showed that prevalence of culture-positive tuberculosis decreased substantially (10.0% per annum, 95% CI: 2.8–16.6%) owing to DOTS after only slight declines related to temporal trends (2.1% annual decline, 95% CI: 1.1–3.2%) and short-course chemotherapy (1.5% annual decline, 95% CI: �9.7% to 11.5%). Under DOTS, the proportion of total cases identified through clinical care increased from 81 to 92%. Conclusions: Following DOTS implementation, prevalence of culture-positive tuberculosis decreased rapidly following a gradual decline for the previous 30 years. In the absence of a large HIV epidemic and with relatively low levels of rifampicin resistance, DOTS was associated with rapid reduction of tuberculosis prevalence

Standardized methodology for drug susceptibility testing of second line drugs is vital for treatment of multi/extensively drug resistant tuberculosis. Discrepancy between laboratory methods and clinical interpretation is well established for bacteriostatic drugs such as ethionamide. Optimization of the standard proportion sensitivity testing (PST) method for ethionamide was under taken in 235 Mycobacterium tuberculosis isolates from new and previously treated pulmonary tuberculosis patients. An additional higher concentration of 57 μg/ml was evaluated against at the standard 40 μg/ml concentration in PST method. Performance parameters and agreement between the two drug concentrations was higher indicating the efficiency of PST method at its present format at 40 μg/ml and additional higher concentration of 57 μg/ml as an alternative when required.

Objective To do a comparative homology search for the 32 mycobacteriophages. Methods This can be estimated as 3 400 proteins from 32 mycobacteriophages assuming each phage has 80-100 genes. The algorithm most widely used for homology detection in comparative genomics is Basic Local Alignment Search Tool (BLAST). Usually a stringent score cutoff is applied to distinguish putative homolog's from possible false positive hits. As a consequence, some BLAST hits are discarded or put into insignificant hits that are in fact homologous. Assigning function to protein sequences is important. Here, we review the status of sequence (BLAST) based and domain based approaches to proteins that can provide functional insights such as database on Protein Families (PFAM). Results The findings showed that 31% of proteins showed similarities with homologous protein with known function. Conclusions In the present study only 31% of mycobacteriophage proteins functions were able to be predicted. Only for 6 proteins, the template structures were available but then they were not directly involved in phage lifecycle. Hence this emphasizes the importance of exploring the structures for mycobacteriophage proteins in order to understand their function and evolutionary significance. Ab initio methods for protein structure prediction can be only alternative for the rest of the proteins but accuracy of prediction is not highly dependable.

Ten extensively drug-resistant tuberculosis (XDR-TB) patients were identified among 104 human immunodeficiency virus negative multidrug-resistant tuberculosis (MDR-TB) patients treated at the Tuberculosis Research Centre, Chennai, India, in two different cohorts between 1999-2003 and 2006-2007. They were managed with individualised treatment regimens. At the time of diagnosis of MDR-TB, one patient had XDR-TB and three had initial ofloxacin resistance. One patient who had had a lobectomy in addition to chemotherapy became bacteriologically negative, three died, three defaulted and the remaining three, who are bacteriologically positive, are still continuing treatment. Although based on a small number of patients, our results have not been encouraging.

Settings. National Institute for Research in Tuberculosis, Chennai.Objective. To assess the proportion of metabolically active cells ofMycobacterium tuberculosisafter exposed to CPC using FDA-EB vital staining and viable counts on LJ medium. Mycolic acid content inM. tuberculosisafter exposure to CPC was estimated using HPLC.Methods. Clinical isolates ofM. tuberculosisand standard reference strainM. tuberculosisH37Rv were used for FDA-EB, viable count, and HPLC.Results. FDA/EB consistently stained 70–90% of log phase cells as green and the remaining cells as red-orange. After CPC treatment, 65–70% of the cells stained red-orange. The viability counts were comparable to 0-day controls. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms.Conclusion. The cells are metabolically inactive during storage with CPC but these cells grew well on LJ medium after removal of CPC. The viability ofM. tuberculosiswas maintained in CPC with minimal reduction. Mycolic acid content was reduced if the cells ofM. tuberculosiswere treated with CPC for 7 days. All the above findings provide yet another evidence for the damage of cell wall ofM. tuberculosis.

Background Shortening tuberculosis (TB) treatment duration is a research priority. This paper presents data from a prematurely terminated randomized clinical trial, of 4-month moxifloxacin or gatifloxacin regimens, in South India. Methods Newly diagnosed, sputum-positive HIV-negative pulmonary TB patients were randomly allocated to receive gatifloxacin or moxifloxacin, along with isoniazid and rifampicin for 4 months with pyrazinamide for first 2 months (G or M) or isoniazid and rifampicin for 6 months with ethambutol and pyrazinamide for first 2 months (C). All regimens were administered thrice-weekly. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The Data and Safety Monitoring Board recommended termination of the trial due to high TB recurrence rates in the G and M regimens. Results Of 416 patients in intent-to-treat analysis, 6 (5%) of 124, 2 (2%) of 110 and 2 (2%) of 137 patients with drug-susceptible TB in the G, M and C arms respectively had unfavorable response at the end of treatment; during the next 24 months, 17 (15%) of 115, 11 (11%) of 104 and 8 (6%) of 132 patients respectively, had TB recurrence. Of 38 drug-resistant patients 1 of 8 and 3 of 26 in the G and C arms respectively had unfavourable response at the end of treatment; and TB recurrence occurred in 2 of 7 and 2 of 23 patients, respectively. The differences in TB recurrence rates between the G and C arms was statistically significant (p = 0.02). Gastro-intestinal symptoms occurred in 23%, 22% and 9% of patients in the G, M and C arms respectively, but most reactions were mild and manageable with symptomatic measures; 1% required regimen modification. Conclusions 4-month thrice-weekly regimens of gatifloxacin or moxifloxacin with isoniazid, rifampicin and pyrazinamide, were inferior to standard 6-month treatment, in patients with newly diagnosed sputum positive pulmonary TB. Trial Registration Clinical Trials Registry of India CTRI/2012/10/003060

One hundred and twenty-seven of 130 isolates of Mycobacterium tuberculosis, suspended in phosphate buffered saline (PBS) and stored at ambient conditions in the laboratory for 14days, and another 55 of 60 cultures, suspended as above, transported from reference laboratories within 7days, were successfully retrieved on LJ medium. Considering the maximum retrieval of M. tuberculosis, use of PBS can be explored further for transportation of M. tuberculosis cultures across laboratories.

To get insight into the sensitivity of fluorescence microscopy for diagnostic and follow-up sputum samples from pulmonary tuberculosis patients in clinical trials, the yield of smear positivity – among culture positive sputum samples – encountered in diagnostic and follow-up samples was retrospectively analyzed from the data available in a mycobacteriology laboratory in India. The sensitivity of fluorescence microscopy for diagnostic and follow-up samples respectively was found to be 94.3% and 60.7%. With these values as guidelines, the performance of fluorescence microscopy in the treatment of multi-drug resistant tuberculosis under DOTS plus program remains to be monitored and studied.

BACKGROUND: Existing methods for the preparation of panel slides necessitate handling high-grade acid-fast bacilli positive sputum samples. OBJECTIVE: To compare panel slides prepared using the phenol ammonium sulphate sediment (PhAS) method with those prepared using the N-acetyl-L-cysteine (NALC) method in proficiency testing. METHODS: Pooled sputum specimens of known smear-positives and -negatives were divided into two parts: one part was used for preparing panel slides using the NALC method and the other using PhAS, a non-hazardous method. Respectively 413 and 384 smears of different grades were prepared in three batches using the PhAS and NALC methods. Smear grade and quality were recorded by 121 microscopists during proficiency testing in different states. Agreement between reference and reported results was analysed using the kappa test. RESULTS: The overall agreement was 96% for the PhAS method and 91% for the NALC method. There were 37 errors using the NALC method compared to 21 for the PhAS method (P

Tuberculosis (TB) is a major public health problem among the Saharia, a marginalized tribal group in Madhya Pradesh state, central India. However, there is no information on the risk factors associated with the development of TB disease in this community. A cross-sectional TB prevalence survey was conducted among the Saharia residing in Sheopur district of Madhya Pradesh. Information on tobacco smoking and alcohol consumption was collected from all the individuals. Persons aged ≥45 years, males, smokers and alcohol consumers had higher risks of developing TB disease. There is an urgent need to develop and implement culturally appropriate awareness raising activities to target smoking and alcohol consumption to support the efforts to control TB in this community.

Background Comparative genomic analysis of M. tuberculosis H37Rv and M. bovis BCG have shown that 16 RDs (Regions of Differences) are deleted in BCG and have shown six deletion regions in M. tuberculosis H37Rv. RD1, is present in M. tuberculosis but is absent in all M. bovis BCG sub-strains. A study from Kerala, a south-western coastal state of India aimed to find out differences in RD1 region showed for the first time the presence of moaA3 gene in majority of their clinical isolates, that was absent in type strain H37Rv. We attempted to find out such polymorphism between type strains and the clinical isolates within RD1, targeting moaA3 gene among the clinical isolates of Tamil Nadu & Pondicherry, south-eastern coastal states of India Methods One hundred and sixteen clinical isolates of M. tuberculosis were included in the study. PCR using RD1DLa and RD1DRa primers was carried out to amplify a 652 bp fragment, encoding for cfp10 and esat 6 proteins of RD1. A second PCR using primers designed from the surrounding regions of moaA3 gene was done to confirm the presence of the full Open Reading Frame (ORF) in clinical isolates. Results In M. tuberculosis H37Rv the expected 652 bp band was present. In BCG it was absent as expected, but a 386 bp fragment was amplified. Around 12/116 (10.3%) of our clinical isolates showed both 652 and 386 bp fragments. The additional 386 bp amplicon is a part of the moaA3 gene which codes for molybdopterin cofactor protein A in M. bovis. The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment. However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates. Conclusion This finding showed that there was regional variation presenting polymorphism in moA3 gene, among the strains of M. tuberculosis and further strengthens the speculation of genetic differences among the strains of Kerala and Tamil Nadu & Pondicherry, the South Indian states

This was a prevalence survey of pulmonary tuberculosis (PTB) disease in the tribal population of Madhya Pradesh state, central India.A community-based cross-sectional tuberculosis (TB) disease prevalence survey was undertaken among adults agedor = 15 years in the tribal population of Madhya Pradesh. A multistage stratified cluster sampling was adopted. A representative random sample of villages predominated by tribal populations was selected from 11 districts. All eligible individuals were questioned for chest symptoms relating to TB. Sputum samples were collected from all eligible individuals, transported to the laboratory, and examined by Ziehl-Neelsen (ZN) smear microscopy and solid media culture methods.Of the 23,411 individuals eligible for screening, 22,270 (95.1%) were screened for symptoms. The overall proportion of symptomatic individuals was 7.9%. Overall prevalence (culture and/or smear positive) of PTB was 387 [95% confidence interval (CI): 273-502] per 100,000 population. The prevalence increased with age and was also significantly higher among males (554/100,000; 95% CI: 415-693) as compared with females (233/100,000; 95% CI: 101-364) (P0.001).The findings suggest that the TB situation amongst the tribal population is not that different from the situation among the non-tribal population in the country. However, TB remains a major public health problem amongst the tribal population and there is a need to maintain and further strengthen TB control measures on a sustained and long-term basis.

Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB) control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP) and previously treated (PT) pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs) in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH), rifampicin (RMP), ofloxacin (OFX) and kanamycin (KAN). Human Immunodeficiency Virus (HIV) status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB) was highest in the treatment failure (35%) group, followed by relapse (13%) and treatment after default (10%) groups. Extensively drug resistant TB (XDRTB) was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.

Assessment of 12 microscopy centers in a tuberculosis unit by blinded checking of eight sputum smears selected by using a lot quality assurance sampling (LQAS) method and by unblinded checking of all positive and five negative slides, among the slides examined in a month in a microscopy centre, revealed that the LQAS method can be implemented in the field to monitor the performance of acid-fast bacillus microscopy centers in national tuberculosis control programs.

Growth of contaminating organisms along with Mycobacterium tuberculosis on Lowenstein-Jensen (LJ) medium is common. However, there is no documented evidence on the decontamination procedure adopted in mycobacteriology laboratories to recover M. tuberculosis from the contaminants grown on LJ medium. At the National Institute for Research in Tuberculosis, of 1048 LJ slopes with M. tuberculosis received from intermediate reference laboratories, 98 (9%) were contaminated. Of these, 87 (89%) M. tuberculosis cultures were retrieved after decontamination with 1% cetrimide. The use of cetrimide as a decontaminating agent to retrieve M. tuberculosis cultures grown with contaminants is documented.

Objective/background: Collection of one spot and one morning sputum specimen is recommended for tuberculosis (TB) drug resistance surveys. This was a retrospective analysis of Mycobacterium tuberculosis cultures isolated from two spot sputum specimens collected from smear positive TB patients in a TB drug resistance survey. It was conducted to understand the value of a second specimen. Methods: A TB drug resistance survey was conducted in the state of Tamil Nadu, India, to estimate the prevalence of drug resistance among new sputum smear-positive (NSP) and previously treated (PT) patients diagnosed in Revised National Tuberculosis Control Program microscopy centers. A total of 2425 patients (1524 NSP and 901 PT cases) were enrolled in the study. From these patients, two spot sputum specimens (C and D) were collected within a period of 2 h. No preservative was added to sputum. The samples were transported at ambient conditions without cold storage to the central laboratory for culture of M. tuberculosis. Culture yield from each sample was computed and analyzed. Results: The proportion of cultures retrieved from C and D specimens among NSP cases (89.3% and 89.7%) and PT cases (90.8% and 90.3%) were similar. The culture grades of C and D samples were comparable (chi-square test, 3560.135; p